Advanced Environmental Biotechnology II

Week 14 - Gene cloning - gene libraries and the selection of

clones

The story so far ….

The environment is made and maintained by living things (organisms).

Organisms can be used to make the environment healthier.

Organisms are chemical factories that take materials and energy in and transform them.

Organisms are made of cells.

Enzymes do the work of cells.

Enzymes are made of proteins, and sometimes RNA.

Proteins and RNA are made of smaller subunits.

Proteins are made of 20 different amino acids arranged in order.

DNA has a code which says which amino acids go in what order to make an enzyme.

The DNA is made of long strings of smaller subunits.

In many microorganisms the DNA is kept in chromosomes.

Some DNA is also found in smaller pieces not in the chromosome.

These smaller pieces are called plasmids.

Plasmids can replicate.

Plasmids can move from one microorganisms to another.

The plasmids also move their DNA, and the codes on the DNA.

Plasmids can be used to carry DNA codes into microorganisms.

These plasmids transform the microorganisms.

The application of genomics and derivative technologies yields insight into ecosystems. The use of genomics, functional genomics, proteomic and systems modeling approaches allows for the analysis of community population structure, functional capabilities and dynamics. The process typically begins with sequencing of DNA extracted from an environmental sample, either after cloning the DNA into a library or by affixing to beads and direct sequencing. After the sequence is assembled, the computational identification of marker genes allows for the identification and phylogenetic classification of the members of the community and enables the design of probes for subsequent population structure experiments. The assignment of sequence fragments into groups that correspond to a single type of organism (a process called ‘binning’) is facilitated by identification of marker genes within the fragments, as well as by other characteristics such as G+C content bias and codon usage preferences. Computational genome annotation, consisting of the prediction of genes and assignment of function using characterized homologs and genomic context, allows for the description of the functional capabilities of the community. Knowledge of the genes present also enables functional genomic and proteomic techniques, applied to extracts of protein and RNA transcripts from the sample. These latter studies inform systems modeling, which can be used to interpret and predict the dynamics of the ecosystem and to guide future studies. qPCR, quantitative polymerase chain reaction.

Molecular approaches for microbial community analysis

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Molecular approaches for microbial community analysis

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Today we will look at how we can use plasmids to transform microorganisms.

These microorganisms can then be grown in clones.

Each clone will have a unique new piece of DNA.

The clones can be grown to make libraries of DNA.

Restriction enzymesRestriction enzymes are proteins which

cut DNA.

They cut DNA whenever a specific DNA sequence is present.

For example, the enzyme called HaeIII cuts at GGCC.

The enzyme EcoRI cuts at GAATTC.

Different restriction enzymes cut at different DNA sequences.

Sticky ends

Some restriction enzymes cut across strands of the DNA molecule to produce overhanging, "sticky" ends.

These sticky ends are useful to join together different DNA molecules.

Res_enz.mov

Restriction Enzymes

3. Examples of the DNA sequences that are recognized by other restriction enzymes are shown below.

HaeIII TaqI5’ – G G C C – 3’ 5’ – T C G A – 3’3’ – C C G G – 5’ 3’ – A G C T – 5’

PstI NotI5’ – C T G C A G – 3’ 5’ – G C G G C C G C – 3’3’ – G A C G T C – 5’ 3’ – C G C C G G C G – 5’

Restriction Enzymes come from Bacteria

Restriction enzymes are used by bacteria to protect themselves against viruses.

They restrict the growth of invading viruses by cutting up the DNA of the virus.

Their names come from the bacteria in which they were discovered.

EcoRI was found in Escherichia coli.

TaqI was found in Thermus aquaticus, a species of bacterium that is found in hot springs.

DNA Ligase

DNA ligase is an enzyme that can join (ligate) DNA molecules together.

Restriction enzymes and DNA ligase are used to clone DNA.

Cutting and ligating DNA

Strategies and steps in cloning.

Basic Steps -1

Cut the vector DNA with a restriction enzyme.

Cut the DNA that we want to clone with the same restriction enzyme.

Mix together the vector DNA with the other DNA.

Add DNA ligase to ligate the DNA molecules together.

The "sticky ends" help in joining the molecules together with DNA ligase.

Basic Steps -2

Put these recombinant DNA molecules into E. coli.

The vector will “transform” the bacterium to become resistant to the antibiotic ampicillin. This is called transformation.

Bacteria with antibiotic resistance have been transformed with the vector and carry a plasmid.

Basic Steps -3

Find the bacteria that carry recombinant plasmids, i.e. plasmids that have become combined with another DNA molecule.

This produces a collection of bacteria that contain fragments of new DNA. This is called a library of cloned DNA.

The basic steps in gene cloning

DNA extracted from an organism known to have the gene of interest is cut into gene-size pieces with restriction enzymes.

Bacterial plasmids are cut with the same restriction enzyme.

The gene-sized DNA and cut plasmids are combined into one test tube. Often, a plasmid and gene-size piece of DNA will anneal together forming a recombinant plasmid (recombinant DNA).

Recombinant plasmids are transferred into bacteria.

The bacteria are plated out and grow into colonies. All the colonies on all the plates are called a gene library.

The gene library is screened to identify the colonies containing the genes of interest by looking for one of three things: the DNA sequence of the gene of interest or a very

similar gene the protein encoded by the gene of interest a DNA marker whose location has been mapped close

to the gene of interest

gene_cloning_in_bac.mov

plasmid_cloning.mov

http://www.whfreeman.com/lodish4e/con_index.htm?99vos

Libraries of Genes

More and more genes are being catalogued (cloned, DNA sequence determined, and filed) from a variety of different sources.

Many bacterial genomes have been sequenced.

A few eukaryote genomes, including human, have also been sequenced.

It is possible to use the internet to look collections of genes that have been cloned from several organisms, and find the functions of those genes.

Gene Libraries - Library Construction

A gene library can be defined as a collection of living bacteria colonies that have been transformed with different pieces of DNA that is the source of the gene of interest.

If a library has a colony of bacteria for every gene, it will consist of tens of thousands of colonies or clones.

Screening the Library

The library must be screened to discover which bacterial colony is making copies of which gene.

The scientist must know either the DNA sequence of the gene, or a very similar gene, the protein that the gene produces, or a DNA marker that has been mapped very close to the gene.

Library screening identifies colonies, which have particular genes.

Growing more Plasmids

When library colonies with the desired genes are located, the bacteria can be grown to make millions of copies of the recombinant plasmids that contain the genes.

Clones

Large insert clonesYACs (Yeast Artificial Chromosomes

Useful for mapping ~1mb insertsUnstable during construction and propagationNot useful for sequencing

BACs (Bacterial Artificial Chromosomes)~150kb insertExtremely stable and easy to propagateGold standard for sequencing targets and chromosome-scale

maps

Cosmids~50kb insertExtremely stable and easy to propagateUseful for sequencing but too small for chromosome maps

Sequence-ready clones

Plasmids1-10kb insert capacityHigh copy numberEasy to sequence bi-directionallyAutomated clone picking/DNA isolation possibleExamples: pUC18, pBR322

Single-stranded Bacteriophage1-5kb insert capacityGrows at high copy as plasmid and is shed into medium as single stranded DNA

phageEasy to isolate, pick, sequenceEasy to automateM13 is used almost exclusively

Microbiological techniques are often based on isolation of pure cultures and morphological, metabolic, biochemical and genetic assays.

They have given lots of information on the biodiversity of microbial communities.

We don’t know enough about the needs of microorganisms.

We don’t know enough about the relationships between organisms.

So we can’t get pure cultures of most microorganisms in natural environments.

Most culture methods are good for certain groups of microorganisms, but other important groups do not live well.

We can use molecular biology approaches.

The techniques are based on the RNA of the small ribosomal subunit or their genes.

Lots of this molecule are found in all living things.

It is a highly conserved molecule but has some highly variable regions.

We can compare organisms, and find the differences.

The gene sequence can be easily sequenced.

In wastewater treatment, microbial molecular ecology techniques have been used mainly to the study of flocs (activated sludge) and biofilms that grow in aerobic treatment systems (trickling filters). This lecture will look at some of those techniques.

Cloning and sequencing the gene that codes for 16S rRNA is the most widely used method.

Nucleic acids are extracted.

The 16S rRNA genes are amplified and cloned.

The genes are sequenced.

The sequence is identified using phylogenetic software.

If we use DNA extracts from microbial communities, the cloning step has to be included.

This is needed to separate the different copies of 16S rDNA. A mixed template cannot be sequenced.

There are over 240,000 sequences deposited in the 16S rDNA NCBI-database.

Half belong to non-cultured and unknown organisms, which were found by 16S rDNA

cloning.

Cloning takes lots of time and so it is not good for analyzing larger sets of samples.

For example, it is not good for looking for changes in natural or engineered microbial communities over time.

Outline of the cloning procedure for studying a microbial community.

(A) Direct nucleic acid extraction, without the need for previous isolation of microorganisms.

(B) amplification of the genes that code for 16S rRNA by polymerase chain reaction (PCR), commonly using universal primers for bacteria or archaea

(C) cloning of the PCR products into a suitable plasmid and transformation of E. coli cells with this vector

(E) selection of transformed clones with an indicator contained in the plasmid (the white colonies) and extraction of plasmid DNA

(F) sequencing of the cloned gene, creating a clone library

(G) Finding the relationships between the cloned sequences of the organisms with the help of computer programs and databases

http://rdp.cme.msu.edu/

The Ribosomal Database Project (RDP) provides ribosome related data and services to the scientific community, including online data analysis and aligned and annotated Bacterial small-subunit 16S rRNA sequences.

Cloning Advantages

Complete 16S rRNA sequencing allows:very precise taxonomic studies and phylogenetic trees

of high resolution to be obtained;design of primers (for PCR) and probes (for FISH).

If time and effort is available, the approach covers most microorganisms, including minority groups, which would be hard to detect with genetic fingerprinting methods.

Cloning DisadvantagesVery time consuming and laborious, making it

unpractical for high sample throughput.Extraction of a DNA pool representative of the

microbial community can be difficult when working with certain sample types (e.g. soil, sediments).

Many clones have to be sequenced so that most of individual species in the sample are covered.

Identification of microorganisms that have not been yet cultured or identified is difficult.

It is not quantitative. The PCR step can favor certain species due to differences in DNA target site accessibility.

Examples of use of clones

Examples of the use of cloning

Find the phylogenetic position of filamentous bacteria in granular sludge.

Find the prevalent sulfate reducing bacteria in a biofilm.

The microbial communities residing in reactors for treating several types of industrial wastewater.

The microbial composition and structure of a rotating biological contactor biofilm for the treatment of ammonium-contaminated wastewaters.

A description of the microbial communities responsible for the anaerobic digestion of manure in continuously stirred tank reactors (CSTR)

Library Area 3, Deep %

Area 3, Shallow %

Area 2% Total %

Number of clones sequenced 960 864 864 Sequences generated 1,920 1,728 1,728 Quality sequences

a1,394 100 1,118 100 1,509 100 4,021 100

Sequences that form contigs 370 26.5 152 13.6 141 9.3 663 16.5 Number of contigs assembled 101 53 54 208 Sequences with similarities to known proteins

b928 66.6 692 61.9 990 65.6 2,610 64.9

Highest similarity to bacterial proteins 901 64.6 629 56.3 890 59.0 2,420 60.2 Highest similarity to Deltaproteobacteria proteins 35 2.5 23 2.1 155 10.3 213 5.3 Highest similarity to archaeal proteins 12 0.9 43 3.8 79 5.2 134 3.3 Highest similarity to eukaryotic proteins 12 0.9 18 1.6 21 1.4 51 1.3

a. Sequences >400nt in length b. e-values <1e-10 from BLASTX searches against the NCBI protein database

Statistics on amplified metagenome library end-sequences

Environmental Whole-Genome Amplification To Access Microbial Populations in Contaminated Sediments

• Recovery of adequate amounts of DNA for molecular analyses can often be challenging in stressed microbial environments.

• Developed multiple displacement amplification (MDA) methods for unbiased, isothermal, amplification of DNA

• Subsequently applied these technologies to understand stressed, low biomass, populations in multiple sediments contaminated with Uranium on the Oak Ridge Reservation

• Over 4000 clones were end sequenced. 5% of all clones were identified as belonging to Deltaproteobacteria (primarily, Geobacter and Desulfovibrio-like)

• Significant overabundance of proteins (COGs) associated with: 1) Carbohydrate transport & metabol. 2) Energy production & conversion, 3) Postranslational modification, protein turnover, & chaperones. --- All of which may be important in adaptation to environmental stressors such as low pH, high contaminate loads, and oligotrophic nature of the subsurface environment

Abulencia, C.B., Wyborski, D.L., Garcia, J., Podar, M., Chen, W., Chang, S. H., Chang, H.W., Watson, D., Brodie, E.L., Hazen, T.C. and Keller, M. (2006) Environmental Whole-Genome Amplification to Access Microbial Populations in Contaminated Sediments. Appl. Environ. Microbiol. 72(5):3291-3301 [download pdf]

Metagenomic Analysis of NABIR FRC Groundwater Community

Metagenomic sequencing:

Almost like a mono-culture52.44 Mb raw data assembled into contigs totaling ~5.5 Mb

224 scaffolds (largest 2.4 Mb)

Genes important to the survival and life style in such environment were found

Extremely low diversity:

Dominated by Frateuria-like organism

At least 2 Frateuria phylotypes

Azoarcus species: less abundant

These results suggest that contaminants have dramatic effects on the groundwater microbial communities, and these populations are well adapted to such environments.

Data: Jizhong Zhou et al.

Phylogenetic Tree of SSU rRNA Genes

BFXI386 AY622233 NABIR FRC soil clone --Reardon

DQ125888 NABIR FRC soil clone --Brodie 4000601 Contig2585 16SrRNA

DQ125806 NABIR FRC soil clone --Brodie

FRC Gamma Group I (87.4%)

AY218719 Uncultured bacterium clone KD78 AY218686 Uncultured bacterium clone KD81

AY188295 Uncultured bacterium clone KD11 AJ010481 Frateuria aurantia

AY495957 Frateuria WJ64 AB100608 Rhodanobacter fulvus AF039167 Rhodanobacter lindaniclasticus L76222 Rhodanobacter lindaniclasticus

BFXI433 AJ583181 uncultured russian disposal site clone

DQ125572 NABIR FRC soil clone --Brodie OR1-87 NABIR FRC soil isolate --Bollmann

DQ125555 NABIR FRC soil clone --Brodie OR1-92 NABIR FRC soil isolate --Bollmann

FRC Gamma Group II (1.6%)

OR1-113 NABIR FRC soil isolate --Bollmann BFXI385

AM084888 uranium mining waste pile clone AJ012069 Herbaspirillum G8A1

AJ505863 Herbaspirillum sp PIV341 Y10146 Herbaspirillum seropedicae

AF164065 Herbaspirillum seropedicae

FRC Beta Group II (4.7)

BFXI398 AY662003 NABIR FRC groundwater clone --Fields

AF408965 Burkholderia str. Ellin123 AF408997 Burkholderia str Ellin155 AF408977 Burkholderia str Ellin135 AF408962 Burkholderia str Ellin120

FRC Beta Group I (3.1%)

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•Four major groups were observed.

•These microorganisms were also found in other studies in this site

Data: Jizhong Zhou et al.Terry Hazen et al.